Dysregulated signaling networks in HNSCC: novel mechanism-based approaches for HSNCC prevention and treatment There is an urgent need for new treatment options for HNSCC patients, considering that their overall 5-year survival is relatively low (50%) and has improved only marginally over the past 3 decades. The emerging knowledge of how the dysregulated function of signaling networks contributes to the initiation, malignant growth, and metastasis of HNSCC can now be exploited to identify novel mechanism-based anti-cancer treatments. Co-targeting mTOR inhibition overcomes cetuximab resistance in HNSCC harboring PIK3CA and RAS mutations. Cetuximab, a monoclonal blocking antibody against the epidermal growth factor receptor EGFR, has been approved for the treatment of HNSCC. However, only few patients display a long-term response to cetuximab, which has prompted the search for mechanisms underlying cetuximab resistance and new therapeutic options to enhance cetuximab effectiveness in HNSCC. We have recently developed an experimental model to study cetuximab resistance in vivo, consisting in retro-engineering cetuximab-sensitive HNSCC cells to express PIK3CA and RAS oncogenes, both of which are identified in HNSCC lesions and can bypass EGFR growth promoting signaling. Cetuximab treatment of PIK3CA- and RAS-expressing HNSCC xenografts promoted an initial anti-tumor response, but all tumors relapsed within few weeks. In these tumors, cetuximab did not decrease the activity of mTOR, a downstream signaling target of EGFR, PIK3CA, and RAS. The combined administration of cetuximab and mTOR inhibitors exerted a remarkably increased anti-tumor activity, particularly in HNSCC cells that are resistant to cetuximab as a single agent. This study revealed that PIK3CA and RAS mutations and other genetic and epigenetic alterations affecting the mTOR pathway may predict the potential resistance to cetuximab. This information can now be also used to select the patients that may benefit the most from the concomitant administration of cetuximab and PI3K and/or mTOR inhibitors as a precision molecular therapeutic option for HNSCC patients. Oral-specific animal models to evaluate molecular-targeted chemopreventive strategies: There is an imperative need to develop effective treatment options to prevent the development of HNSCC in patients harboring potential pre-malignant lesions. The use of a novel oral premalignancy model revealed a remarkable chemoprotective activity of metformin, a relatively safe drug used by millions of Americans for type II diabetes. In a recent study we have shown that metformin reduces mTOR activity in oral premalignant lesions through stimulation of the AMP-activated protein kinase (AMPK) signaling pathway. We have optimized the oral delivery of metformin to achieve clinically relevant blood levels, and explored its preclinical activity in representative HNSCC tumor xenograft models. HNSCC cells harboring PIK3CA mutations or HPV-oncogenes were very sensitive to the growth inhibitory activity of metformin in vitro, concomitant with AMPK activation and mTOR inhibition. These effects were dependent on the expression of organic cation transporter 3 (OCT3), a metformin uptake transporter that is highly expressed in dysplastic oral squamous epithelium and HNSCC. Furthermore, metformin administration in vivo caused a dramatic, OCT3-dependent reduction in tumor growth of PIK3CA and HPV+ HNSCC cells implanted into immunocompromised mice. In this regard, the incidence of HPV-related malignancies, including HNSCC, is increasing among HIV-infected individuals, irrespective of the restoration of their immune function by the use of highly active antiretroviral therapy. This prompted us to examine mTOR activation and OCT3 expression as surrogate biomarkers for metformin sensitivity, in a large panel of HNSCC lesions from HIV- and HIV-infected (HIV+) individuals (490 total). We found that the percentage of HNSCC cases that were HPV+ is significantly higher in the HIV+ population compared to the HIV- population. The majority of these lesions, as well as perianal and cervical SCCs arising in HIV+ patients, exhibit increased mTOR activity and high OCT3 expression. Our study supports that the PI3K-mTOR pathway is a widespread event in HNSCC, including HPV- and HPV+ lesions arising in HIV+ patients, all of which express OCT3. These observations may provide a rationale for the clinical evaluation of metformin to halt SCC development from precancerous lesions, including in HIV-infected individuals that are at high risk of developing HPV-associated cancers. Genomic and proteomic approaches to understand oral cancer We have conducted gene and protein expression analysis of HNSCC. These efforts are providing a wealth of information about the dysregulated molecular circuitries driving HNSCC development, hence facilitating the identification of new therapeutic targets and suitable biomarkers for monitoring HNSCC progression and treatment response. Exploiting the head and neck cancer oncogenome. The recent development of deep sequencing approaches to study human cancer genomes in individual tumor lesions is already revolutionizing medical oncology and translational medicine. This large and growing body of information is now contributing to the elucidation of aberrant molecular mechanisms driving tumor progression, hence revealing novel druggable targets for therapeutic intervention to prevent and treat human cancers. The emerging picture is that despite the remarkable complexity of genomic alterations found in HNSCC, most of them fall within few major driver-signaling pathways, with the majority of the HNSCC lesions harboring genetic and epigenetic alterations that converge on the persistent activation of the PI3K-mTOR pathway. While representing a major HNSCC driver, this likely overreliance on the PI3K-mTOR signaling route for tumor growth can in turn expose a cancer vulnerability that can be exploited for therapeutic purposes. Indeed, the have documented the sensitivity of HNSCC to mTOR inhibition in multiple experimental models and encouraging recent clinical studies. The presence of genomic alterations in the PI3K pathway may also represent a suitable biomarker predicting a clinical response to its pharmacological inhibitors. Semaphorin 3F is a HNSCC anti-lymphangiogenic metastasis suppressor gene. HNSCC metastasize primarily to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCC demonstrate high intratumoral lymphangiogenesis that correlates with lymph node invasion and metastasis. In this regard, the gene for SEMA3F, a member of the semaphorin family of axon guidance molecules is localized to 3p21, one of the earliest and most frequently deleted loci in HNSCC. We have shown that semaphorins can control normal and tumor angiogenesis, and hence hypothesized that SEMA3F may function in HNSCC progression. In a recent study, we demonstrated that heterozygous deletion and decreased expression of SEMA3F correlates with poor clinical outcome, increased vascularity, and metastasis. We found that SEMA3F negatively regulates the function of lymphatic endothelial cells and blocks lymphangiogenesis in vivo. SEMA3F signals through multiple Neuropilin/Plexin A co-receptor complexes, though its physiologic role is predominantly dependent on NRP2, a neuropilin that is highly expressed in lymphatic endothelial cells. Remarkably, expression of SEMA3F inhibited intratumoral lymphangiogenesis and blocked lymphatic metastasis. Together, this indicates that SEMA3F functions as an antilymphangiogenic tumor and metastasis suppressor in HNSCC through paracrine signaling pathways. SEMA3F may be a useful biomarker and possible therapeutic target in HNSCC.